The MYC proto-oncogene family plays a central role in the response to mitogenic stimuli and the control of proliferation in normal and malignant cells. c-Myc protein overexpression is a hallmark of Burkitt's lymphoma, and N-MYC amplification is a critical prognostic factor in neuroblastoma. MYC gene expression is also deregulated in many other cancers, but the molecular mechanisms that regulate Myc activity are only partly elucidated. This proposal will specifically investigate the role of Mxii and MxiO, two Myc family members, in modulating the activity of both c- and N-Myc. As a transcription factor, Myc regulates expression of genes related to cell growth, division and apoptosis. In contrast to Myc, Mxii represses transcription of some Myc-regulated genes, counteracting the effects of Myc. Thus, Mxii is a Myc antagonist. We have shown that Mxii expression results in growth arrest, suppressing cell proliferation in vitro. Therefore, we hypothesize that reduced Mxii activity is likely to potentiate Myc-dependent proliferation. We recently identified MxiO, a novel, alternatively transcribed Mxii isoform that lacks the growth suppressive activity of Mxii. While MxiO and Mxii are concomitantly expressed in many cell lines and tissues, the relative levels of MxiO are higher in tumors and tumor cell lines than in normal cells. This variation in levels of MxiO and Mxii suggests that the Myc-inhibitory activity of Mxii may be modulated by MxiO. We postulate that MxiO is an Mxii antagonist. This notion of a single gene giving rise to protein products with alternative biological activities is well established in the case of Bcl-x (Bcl-xL vs. Bcl-xS) and Ink4a (p16 vs. p14). In this proposal, we will explore the interactions of MxiO and Mxil with each other and with Myc, in the context of both cell proliferation and neoplasia, by setting the following Specific Aims: (1) Characterize the biological activity of MxiO; (2) Determine how expression of MxiO and Mxii are coordinately regulated, using our experience with the Mxii promoter to determine the factors that regulate expression through the MxiO promoter; (3) Determine mechanisms of growth regulation by MxiO and Mxii in the context of Myc-we will use models of c-Myc-induced transformation, as well as N-Myc-dependent neuroblastoma proliferation to explore these interactions, and also study patterns of gene expression; and (4) Evaluate the in vivo effects of specifically inactivating mxii or mxiO in transgenic mice. Through these studies, we will gain a better understanding of the role of these Mxii isoforms in the complex Myc signaling pathways involved in cell growth regulation and neoplasia.